TGFBR2-SE, a new soluble human TGF-beta type II receptor isoform, is sumoylated, interacts with cytoplasmic and nuclear proteins, and is secreted on exosomes.

JORGE VELASCO ZAMORA; TANIA RODRIGUEZ; RICARDO DEWEY; MARCELA BERTOLIO; MARCELO PERONE

Mar del Plata

Congreso; LXIII Reunión Científica Anual de la Sociedad Argentina de Investigación Clínica (SAIC); 2018

Sociedad Argentina de Investigación Clínica

We have recently described in human cells a new splicing variant ofTGF-β type II receptor lacking the last 63 nucleotides of exon II and the first86 nucleotides of exon III. This deletion of 149 nucleotides causes aframeshift with the appearance of an early stop codon rendering atruncated protein of 80 amino acids lacking the transmembrane domain, known as TGFBR2 solubleendogenous (TGFBR2-SE). Based on a computational sumoylation analysis we found that TGFBR2-SE has three potential non-concensus sumoylationlysines (K76, 77 and 78). To test whether TGFBR2?SE is actually sumoylated, weperformed protein immunoprecipitationof 293T cell lysates with a specific a-TGFBR2-SE pAb,followed by immunoblotting with mAbs (a-TGFBR2?SE, and a-SUMO1). In this way we found that the new isoform isposttranslationally modified by sumo addition. For further proteincharacterization, and toallow increased expression in mammalian cells, we codon optimized the TGFBR2-SEcDNA. Furthermore, to ease protein purification and enhanced in vivohalf-life, we fused it in frame with the human IgG1 Fc domain, and expressed it in 293T cells by means of alentiviral vector (Lv.TGFBR2-SE/Fc). Additionally, we identified the interactome of purifiedTGFBR2-SE/Fc by screening of the Huprot v3.0 Human Proteome array.This assay indicated that TGFBR2-SE is able to bind to a panel of 155 proteins.This set of proteins was compared with exosomal Vesiclepedia data usingFunRich, indicating that TGFBR2?SE interactswith 79 proteins present in exosomes, of which 67 are found either only in the cytoplasm(22), only in the nucleus (11) or both (34). Finally, we confirm exosomalsecretion of TGFBR2-SE and the Fc-tag protein by Western blot of 293T cellmicrovesicles with mAbs (a-TGFBR2-SEand a-CD63).These results show for the first time important TGFBR2-SE characteristicscontributing to unravel the function of the newly described isoform.